Fig. 4

BMP9 overexpression promotes liver toxicity, multi-organ wasting, and elevated muscle atrophy markers despite BMP-SMAD1/5 signaling. (A) Body weight, blood glucose, and levels of liver damage enzymes: alanine aminotransferase (ALT) and aspartate aminotransferase (AST). (B) Heart, liver and epididymal fat pat weights. (C) Weights of lower limb skeletal muscles: quadriceps, gastrocnemius, tibialis anterior, and soleus. In all graphs, light gray bars represent a control group that received an empty vector via a hydrodynamic tail vein injection (n = 11–12) and dark gray bars represent a group that received 20 ug of BMP9 DNA (n = 14). (D) Impact of hepatic BMP9 over-expression for 7 days on signaling in quadriceps muscles. Immunoblots for phosphorylated SMAD1/5 and phosphorylated SMAD2 and total SMAD1 and total SMAD2/3; phosphorylated ribosomal protein 6 S and total S6, MuRF1 and MAFbx in quadriceps muscles of mice that received DNA with an empty vector (control) or BMP9 DNA (BMP9). Immunoblots for vinculin as shown as protein loading controls. Molecular weights are shown on the left-hand side for each blot and arrows point to the protein bands of interest. (E) Impact of hepatic BMP9 over-expression for 7 days on gene expression in quadriceps and gastrocnemius muscles. mRNA amounts for specific genes were quantified by real-time quantitative polymerase chain reaction and expression levels were normalized to a geometric mean of reference genes. Vertical dotted lines in each graph show mRNA levels for each gene in the control group that received DNA with an empty vector (n = 12). Dark gray bars show mRNA levels for specific genes in the group that received BMP9 DNA (n = 14), relative to the control group. Data are mean ± standard deviation (SD) from the mean. Statistical analyses were conducted with an un-paired t-test. *p ≤.05; **p ≤.01; ***p ≤.001; ****p ≤.0001 compared to control